Molecular mechanisms of regulation by a β‐alanine‐responsive Lrp‐type transcription factor from Acidianus hospitalis

Abstract The leucine‐responsive regulatory protein (Lrp) family of transcriptional regulators is widespread among prokaryotes and especially well‐represented in archaea. It harbors members with diverse functional mechanisms and physiological roles, often linked to the regulation of amino acid metabolism. BarR is an Lrp‐type regulator that is conserved in thermoacidophilic Thermoprotei belonging to the order Sulfolobales and is responsive to the non‐proteinogenic amino acid β‐alanine. In this work, we unravel molecular mechanisms of the Acidianus hospitalis BarR homolog, Ah‐BarR. Using a heterologous reporter gene system in Escherichia coli, we demonstrate that Ah‐BarR is a dual‐function transcription regulator that is capable of repressing transcription of its own gene and activating transcription of an aminotransferase gene, which is divergently transcribed from a common intergenic region. Atomic force microscopy (AFM) visualization reveals a conformation in which the intergenic region appears wrapped around an octameric Ah‐BarR protein. β‐alanine causes small conformational changes without affecting the oligomeric state of the protein, resulting in a relief of regulation while the regulator remains bound to the DNA. This regulatory and ligand response is different from the orthologous regulators in Sulfolobus acidocaldarius and Sulfurisphaera tokodaii, which is possibly explained by a distinct binding site organization and/or by the presence of an additional C‐terminal tail in Ah‐BarR. By performing site‐directed mutagenesis, this tail is shown to be involved in ligand‐binding response.


| INTRODUCTION
The Lrp family of transcription factors, named after the prototypical leucine-responsive regulatory protein (Lrp) from Escherichia coli, is a widespread and abundant family of regulators among prokaryotes, especially for the archaea (Peeters & Charlier, 2010;Perez-Rueda et al., 2018;Yokoyama et al., 2006;Ziegler & Freddolino, 2021). They are also called feast/famine regulatory proteins (FFRPs) (Calvo & Matthews, 1994), referring to the functional role of some family members that regulate the transcription of metabolic genes in response to the availability of nutrients, more specifically amino acids. Other members are involved in the regulation of diverse physiological functions such as transport, antibiotic biosynthesis, motility, DNA repair, and recombination or virulence (Calvo & Matthews, 1994;Chen et al., 2019;Deng et al., 2011;Liu et al., 2017;López-Torrejón et al., 2006). Lrp-type regulators have been found to fulfill either a specific or a global role (Friedberg et al., 2001;Kawashima et al., 2008;Unoarumhi et al., 2016), which relates to their intracellular abundance and the regulon size. Some regulators function not only as transcription regulators but also as nucleoidassociated proteins (NAP), involved in the organization of the chromosome structure (Peterson et al., 2007).
The monomeric structure of an Lrp-type protein is composed of two domains that are connected by a flexible linker. The N-terminal DNA-binding domain (DBD) harbors a helix-turn-helix motif or, in most archaeal members, a winged helix-turn-helix motif (Peeters & Charlier, 2010). The C-terminal effector binding domain (EBD), also named the regulation of amino acid metabolism (RAM) domain (Ettema et al., 2002), is characterized by an αβ-sandwich fold and is of importance for ligand binding and oligomerization of the protein.
Archaeal Lrp-like transcription factors often form higher-order oligomers, mostly octamers, with the DNA being wrapped around the interacting protein (de los Rios & Perona, 2007;Koike et al., 2004;Kumarevel et al., 2008;Reddy et al., 2007;Thaw et al., 2006). Lrptype transcription factors employ different regulatory mechanisms, leading to transcriptional repression and/or activation. Upon interaction with effector molecules, in most cases amino acids, the DNAbinding affinity and/or transcriptional output is altered via an allosteric response (Kawashima et al., 2008).
In contrast to most Lrp-type regulators, which are typically responsive to α-amino acids, a conserved Lrp-type regulator in the thermoacidophilic Thermoprotei Sulfolobus acidocaldarius and Sulfurisphaera tokodaii interacts with and responds specifically to the nonproteinogenic β-amino acid β-alanine (Liu et al., 2014). This protein was named BarR, hereafter referred to as Sa-BarR and St-BarR for the homologs in S. acidocaldarius and S. tokodaii, respectively. The BarR-encoding gene is organized in a conserved divergent operon together with a predicted aminotransferase gene and, in the case of S. tokodaii, adjacent to a semialdehyde dehydrogenase gene. In S. acidocaldarius, Sa-BarR was shown to exert a β-alanine-dependent transcriptional activation of the divergently oriented aminotransferase gene and a β-alanine-independent transcriptional autoactivation of its gene (Liu et al., 2014). This was corroborated by the observation that Sa-BarR interacts with the sa-barR-aminotransferase intergenic region in vivo, irrespective of the presence of β-alanine in the growth medium (Liu et al., 2016). On the contrary, the addition of β-alanine caused dissociation of the Sa-BarR-DNA complex in in vitro experiments (Liu et al., 2014). Sa-BarR and St-BarR form octameric structures and the barR-aminotransferase intergenic region harbors multiple binding sites for the regulator, characterized by a 15-basepair (bp) semipalindromic binding motif. It is hypothesized that each binding site is contacted by a dimeric portion of the protein and that the DNA is wrapped around a BarR octamer during interaction (Liu et al., 2014).
BarR displays a high sequence identity with Grp, a glutamineresponsive Lrp-type regulator in S. tokodaii (Kumarevel et al., 2008). Grp harbors 69% amino acid sequence identity with St-BarR and Sa-BarR and it is therefore assumed that Grp and St-BarR are the result of a gene duplication event in S. tokodaii (Kumarevel et al., 2008). The crystal structure of Grp has been used as a template in the structural modeling of Sa-BarR (Liu et al., 2014). BarR is conserved in other Sulfolobales, including Acidianus hospitalis, a species that also has an acidothermophilic lifestyle, growing optimally at temperatures between 65°C and 95°C and a pH between 2 and 4 (You et al., 2011). A. hospitalis has a facultative anaerobic chemolithoautotrophic metabolism that relies on sulfur. As compared to model species belonging to Sulfolobales, A. hospitalis has not been extensively investigated: only a single protein (Aho7c) has been characterized in detail thus far (Kalichuk et al., 2017).
In this work, the A. hospitalis BarR homolog, referred to as Ah-BarR, is investigated. The DNA wrapping hypothesis is further examined by performing high-resolution contact probing of the interaction between Ah-BarR and its operator DNA and an AFM visualization of the conformation of the formed nucleoprotein complexes. Moreover, transcription regulatory mechanisms are studied in-depth by using a reporter gene system in a bacterial host, and by employing a mutagenesis approach, structural determinants of β-alanine ligand response are identified in Ah-BarR.

| Bioinformatic analyses and structural predictions
Clustal Omega (Madeira et al., 2022;Sievers et al., 2011) was used for protein sequence alignment, and MUSCLE (Edgar, 2004;Madeira et al., 2022) for DNA sequence alignment of intergenic promoter regions. Protein structures were predicted with AlphaFold (Jumper et al., 2021) and homology modeling was done with SWISS-MODEL (Waterhouse et al., 2018). Visualizations of protein structures, as well as structural alignments, were performed in PyMOL (Schrödinger & Delano, 2020). Transcription factor binding sites were predicted using FIMO (Grant et al., 2011). Sequence logos were created with WebLogo 3 (Crooks et al., 2004), using both the forward and reverse sequences. Molecular docking of ligand interaction was done with the program AutoDock Vina (Trott & Olson, 2010).

| Cloning and site-directed mutagenesis
The ah-barR coding sequence (ahos_rs02205) was codon-optimized for E. coli and a construct was designed in which it was fused to linkers containing homology regions to the insertion site on pET24a.
This construct was ordered at Twist Bioscience, as well as a synthetic DNA fragment of gb_Ahos ( Figure A1). Genomic DNA of S.
acidocaldarius was used as a template for amplification of the saci_rs10330-saci_rs10335 region. A detailed overview of the used primers, restriction enzymes, and constructs generated in this work is provided in Tables A1-A3. Cloning of the barR gene, gb_Ahos, or promoters in the different plasmid vectors was performed as described (Bernauw et al., 2022).
PCRs were performed using KAPA HiFi DNA polymerase (Roche), plasmids were restricted using FastDigest restriction enzymes (Thermo Scientific) and all reactions were analyzed by agarose gel electrophoresis and purified using the Wizard ® SV Gel and PCR Clean-Up System (Promega). SLiCE (Seamless Ligation Cloning Extract) method (Zhang et al., 2012) was performed as described (Bernauw et al., 2022), followed by heat shock transformation in competent E. coli DH5α or MG1655 cells. Colony PCR and Sanger sequencing (Eurofins Genomics) were employed to verify the sequences of the constructs.
pACYC184 gb_Ahos with mutated promoter sequences (mut1, mut2, and mut3), as well as pET24a and pITC plasmids containing mutated Ah-BarR sequences (M103A, M103N, M103T, T134A, and T136A) were generated according to the site-directed mutagenesis method described by Edelheit et al. (2009). To this end, PCRs were performed using 500 ng of the restricted wild type (WT) plasmid, 40 pmol of a single primer (either forward or reverse), and KAPA HiFi DNA polymerase (Roche). Corresponding forward and reverse reaction products were combined, followed by a denaturation and annealing step, digestion by the addition of 30 units FastDigest DpnI restriction enzyme (Thermo Scientific), and incubation for 2 h at 37°C. The truncated Ah-BarR mutants were generated by first amplifying the truncated gene using PCR and by restriction with FastDigest restriction enzymes (Thermo Scientific) of the plasmid backbone, followed by Gibson Assembly cloning using NEBuilder ® HiFi DNA Assembly Master Mix (New England Biolabs Inc.). Finally, all mixtures were transformed by heat shock transformation in E. coli DH5α chemically competent cells.

| Protein expression, purification, and size exclusion chromatography
To perform heterologous expression of Ah-BarR WT and mutant proteins, the pET24a ah-barR (WT/mutant) plasmids were first transformed in chemically competent cells of E. coli SoluBL21. A 300 mL culture was grown at 37°C to an optical density (OD 600nm ) of 0.6, after which the cells were induced with 1 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) and further incubated for 16 h at 37°C. Cells were pelleted and resuspended in buffer A (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 40 mM imidazole), followed by the addition of 4 mM Pefabloc ® (Roche) and sonication using a Vibracell 75043 (Bioblock Scientific) at 4°C and 20% of the maximal amplitude during 15 min. Next, lysed cells were centrifuged, and the supernatant was subjected to an additional heat treatment for 10 min at 75°C.
After centrifugation, the remaining supernatant was used for purification of the C-terminally His-tagged Ah-BarR proteins by affinity chromatography using a 1 mL HisTrap FF column (Cytiva), coupled to an ÄKTA FPLC system (Cytiva) equipped with a UPC-900 monitor (Cytiva). Equilibration of the column was done with buffer A, while a linear gradient of 0%-100% buffer B (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM imidazole) was applied over 40 column volumes to elute the His-tagged protein. Eluted fractions were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the Ah-BarR-containing fractions were dialyzed in storage buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl). All proteins were concentrated using Vivaspin ® 2 (MWCO 5000, Sartorius) up to a concentration between 0.7 and 1 mg/mL. SEC-MALS analysis was performed on 30 µL of each protein preparation on a Superdex 200 increase 5/150 GL column (Cytiva), coupled to an HPLC Alliance system (Waters) equipped with a 2998 PDA detector (Waters), a TREOS II MALS detector (Wyatt Technology) and a RI-501 refractive index detector (Shodex). Additionally, 0.7 mg of WT BarR protein was analyzed in the absence and presence of 100 mM β-alanine in an SEC experiment using a HiLoad ® 16/60 Superdex ® 200 prep grade column (Cytiva), coupled to an ÄKTA FPLC system (Cytiva) equipped with a UPC-900 monitor (Cytiva).
Labeled promoter fragments were obtained by PCR using Taq DNA polymerase (Promega), a 32 P-labeled primer, a non-labeled second primer, and a plasmid template (Table A4) GraphPad PRISM (version 9.3.1 for Windows, GraphPad Software, www.graphpad.com) was used to perform Hill curve fitting using nonlinear regression for saturated binding (one site, specific binding), and to determine apparent equilibrium dissociation constants K D .
"In-gel" Cu-OP footprinting was performed as described (Charlier & Bervoets, 2022). First, an EMSA experiment was performed with each reaction containing 300 cps of 32 P-labeled promoter fragment (bottom strand labeled) (Table A4) and an Ah-BarR octameric protein concentration of 0, 108, and 216 nM. After electrophoresis, the gel was immersed in 200 mL of 10 mM Tris-HCl, pH 8.0, followed by the addition of a 20 mL solution, composed of 1 mL 40 mM 1,10phenanthroline (in ethanol), 1 mL 9 mM CuSO 4 and 18 mL of nuclease-free water. After 5 min, 10 mL of 100X diluted 3mercaptopropionic acid was added and 10 min later, 20 mL of a 30 mM neocuproine solution was added. After 5 min of incubation, the gel was rinsed, an X-ray-sensitive film was exposed to the gel for 2 h and the bands corresponding to input DNA and the Ah-BarR-DNA complexes were recovered from the gel. A, T, G, and C ladders were prepared using the USB ® Thermo Sequenase Cycle Sequencing kit (Applied Biosystems). All samples were loaded on a 6% denaturing polyacrylamide gel and electrophoresis was performed. Gels were visualized using a Storage Phosphor Screen BAS-IP MS (Cytiva) and Personal Molecular Imager (PMI) system (Bio-Rad). Densitometry of the footprint pattern was performed using ImageJ (Schneider et al., 2012), after which the ratio of unbound to bound DNA was calculated for each of the individual bands.

| AFM
Before AFM, a PCR was performed using KAPA HiFi DNA polymerase (Roche Diagnostics) to generate the 780 bp operator fragment (Table A4), followed by a purification using the Wizard ® SV Gel and PCR Clean-Up System (Promega). Purified DNA and Ah-BarR protein were diluted in protein-DNA binding buffer to concentrations of respectively 25 nM DNA and 49 nM Ah-BarR (octameric concentration). Equal volumes of DNA and protein were mixed and incubated for 15 min at 37°C, after which 2 µL of the reaction was mixed with 28 µL of adsorption buffer (40 mM HEPES pH 7.1, 10 mM NiCl 2 ). Twenty microliters of this mixture were deposited on a freshly cleaved mica sheet, followed by 10 min of incubation. Subsequently, the mica surface was extensively rinsed with washing buffer (20 mM HEPES pH 7.4, 3 mM NiCl 2 ), after which 200 µL of washing buffer was added. Next, visualization was performed in liquid using an AFM microscope (NanoWizard 4 Ultraspeed 2, Bruker-JPK). Images were acquired using FASTSCAN-D probes (resonance frequencies 80-140 kHz and nominal spring constant of 0.25 N/m, Bruker) in AC Mode Fast Imaging. Scan sizes ranged between 170 × 170 nm and 2 × 2 µm. Images were processed through the JPK Data Processing software and Gwyddion (leveling data, correcting scars, and adapting the color scheme). All images were obtained using the same sample and probe. Images of all complexes are available in the Appendix.

| Reporter gene assays
Reporter gene assays were performed as previously described (Bernauw et al., 2022). pPRC6 and pITC variants (Table 1) containing medium were measured as well to correct for the background signal.
Reporter expression was analyzed at t = 30 h, when all cultures had reached the stationary phase, by taking into account three subsequent time points (t-1, t, t+1). The corrected FL/OD 600 value was calculated for each replicate (n = 4) as follows: Response curves were fitted to a Hill function of the shape according to a previously published procedure (Landry et al., 2018) with minor adaptations. In this equation, P represents the normalized fluorescent output FL OD as a function of M, the β-alanine concentration, b refers to the basal output, and a to the maximum increase in output. The threshold θ represents the β-alanine concentration for which 50% of the maximum output is attained, relative to the basal level and n is the Hill coefficient. Briefly, lmfit 1.1.0 was used to fit all replicates of a response curve to the appropriate Hill function using the Levenberg-Marquardt algorithm (Moré, 1978;Newville et al., 2016). Residuals were weighted by multiplying each residual by the inverse of the mean normalized fluorescence ( )

FL OD
− −1 at the corresponding β-alanine concentration to fit low and high FL OD values equally well. The results of fitting were displayed by plotting the experimental data along with the fitted Hill function using Matplotlib 3.6.2 on a symmetrical logarithmic scale with the linthresh parameter set to 2, which draws up the x-axis with a linear area (from 0 to 2 mM) and a logarithmic area (from 2 to 10 mM) (Hunter, 2007). All best-fit parameters are supplied in Table 2 along with their 95% confidence intervals calculated using the conf_interval function, which performs an F-test. The dynamic range was estimated as μ = a b . Statistical analysis of the FL/OD 600 values was performed in R, using the packages ggplot2, ggpubr, tidyverse, broom, and AICcmodavg. Oneway analysis of variance [ANOVA] was performed, testing the significance of differences between all conditions measured for one biosensor.
After that, a Tukey's HSD (honestly significant difference) test was performed to assess the significance between specific conditions. 3 | RESULTS

| Ah-BarR is an octameric protein with typical Lrp-type structural features
Ah-BarR (AHOS_RS02205) displays amino acid sequence identities of 66%, 63%, and 64% with its homologs Sa-BarR from S. acidocaldarius, St-BarR from S. tokodaii and Grp from S. tokodaii, respectively ( Figure 1a). Upon predicting its monomeric structure in AlphaFold, it was confirmed that Ah-BarR harbors typical structural characteristics of an Lrp-type protein ( Figure 1b). The N-terminal DBD folds into a helix-turn-helix motif, composed of helices α1-α3, while the C-terminal EBD harbors the typical αβ-sandwich fold (β1, α4, β2, β3, α5, and β4). In comparison to Sa-BarR, St-BarR, and Grp, Ah-BarR has an additional C-terminal tail of 13 amino acids (Figure 1a,b). For the remainder of the protein structure, Ah-BarR is predicted to display a high degree of structural similarity with its homologs. Indeed, the Ah-BarR model can be structurally aligned with the Grp crystal structure with an RMSD of 0.751 Å (Figure 1c). Based on the octameric conformation of S. tokodaii St-BarR and Grp (Kumarevel et al., 2008;Liu et al., 2014), the Ah-BarR structure was also modeled as an octamer shown that β-alanine causes dissociation of protein-DNA complexes in vitro (Liu et al., 2014), the addition of 5 mM β-alanine to binding reactions did not significantly alter the formation of Ah-BarR-DNA complexes (Figures 2a and A2).
Next, EMSAs were performed using operator mutant fragments to establish the role of each of the predicted binding motifs TFBS1, BERNAUW ET AL | 5 of 29 TFBS2, and TFBS3 for Ah-BarR binding ( Figure 2b). In these operator mutant fragments, the last three bps of one or more predicted binding sites were mutated (CWR to TTT, with W = A/T and R = A/G), either only in TFBS1 (mut1), both in TFBS1 and TFBS2 (mut2) or in all three sites (mut3). Upon mutating TFBS1 (mut1), complex formation was still observed, albeit with a lowered binding affinity (K D of 31 nM vs. 12 nM for the WT operator) (Figure 2b and A2). For the mut2 operator mutant fragment, the effect on binding affinity was similar as observed for mut1 (K D of 36 nM); however, two complexes were formed instead of one, each with different relative mobility and smearing was observed for the mut2 fragment, pointing to the lower stability of the nucleoprotein complexes causing dissociation during electrophoresis. In the EMSA with the triple mutant (mut3), the binding of Ah-BarR was completely abolished (Figure 2b). These experiments indicate that all three predicted binding sites contribute to complex formation.
To perform a high-resolution contact probing of the interaction between Ah-BarR and the intergenic region, a footprinting experiment was performed (Figure 2c). To this end, a shorter 168-bp fragment of the intergenic region was subjected to an "in-gel" copper-phenanthroline (Cu-OP) footprinting procedure with the bottom strand labeled, enabling to separate the Ah-BarR complexes from unbound DNA (input DNA) for a separate analysis. It was observed that both TFBS 2 and TFBS 3 were specifically protected by Ah-BarR in Ah-BarR-DNA complexes, while the promoter of the aminotransferase gene remained unaffected ( Figure 2c). Contrarily to TFBS 2 and TFBS 3, TFBS 1 did not seem to be protected by Ah-BarR, which can be explained by multiple possible reasons: (i) this binding site is further removed from the 32 P-labeled end of the labeled strand than the other two binding sites, thereby limiting the resolution of the footprint, making it harder to discern protected areas and ii) based on the binding analysis to operator mutant fragments (Figure 2b), it can be hypothesized that TFBS 1 has less favorable binding kinetics (as compared to TFBS 2 and TFBS 3) that are not as easily captured by chemical footprinting (e.g., due to higher dissociation rate). Not only protection but also hyperreactivity zones were observed in the footprinting experiment, especially in the spacer region between TFBS 2 and TFBS 3 (Figure 2c), pointing to the establishment of protein-induced DNA deformations.
The consensus sequence for Ah-BarR binding, based on all three binding site sequences (Figure 2d), displays a high similarity to that of Sa-BarR (Liu et al., 2014), with an AT-rich center and palindromic T A B L E 2 Dose-response parameters for Ah-BarR biosensor strains. Note: Reported values are best-fit values for fits to a Hill function, together with the 95% confidence interval indicated between brackets (see also Section 2.6).
half-sites. Less favorable binding kinetics for TFBS 1 might be explained by the presence of a C-T bp on position 10 in the AT-rich center and/or by an imperfect second-half site (CTA instead of CAA). 3.4 | Transcription regulation by Ah-BarR can be monitored with a heterologous system in a bacterial host

| Architectural conformation of
In contrast to S. acidocaldarius, A. hospitalis is not accessible for genetic experiments. We, therefore, sought an alternative approach to study the mechanisms of transcription regulation and ligand response of Ah-BarR. To this end, a heterologous Ah-BarR-specific reporter gene assay was developed in the model bacterium E. coli. and BS4 using the S. acidocaldarius promoters P sa-barR and P sa-at , respectively, in combination with Ah-BarR. Similar regulatory behavior was observed for BS1 and BS2, although both dose-response curves were characterized by lower threshold values and higher Hill coefficients ( Figure 5, Table 2).

| Structural determinants of β-alanine interaction and response
Based on the interactions of the homolog Grp in S. tokodaii with its ligand glutamine (Kumarevel et al., 2008), in silico docking of β-alanine Although all mutants were still sensitive to β-alanine, differences were notable. Reporter gene experiments in E. coli showed a significantly decreased sensitivity to β-alanine for mutants M103A, T134A, and T136A ( Figure 7). Although this observation indicates that these residues are indeed important for interaction with βalanine, the substitution of M103 for asparagine (M103N) or threonine (M103T), did not significantly alter the β-alanine response.
Moreover, these mutations did not lead to the acquisition of a glutamine-specific response, indicating that ligand-binding specificity F I G U R E 2 In vitro analysis of Ah-BarR binding to the barR-aminotransferase intergenic region. TFBS, transcription factor binding site; at, aminotransferase; F, unbound DNA; S, single-stranded DNA; B, protein-DNA binding complex; W, well. (a) Electrophoretic mobility shift assays (EMSAs) using a 274 bp fragment covering the intergenic region. The Ah-BarR protein concentration is indicated in octameric units. In the left panel, the EMSA is performed in the absence of β-alanine, while in the right panel, a fixed concentration of 5 mM β-alanine was added. Apparent equilibrium dissociation constants K D were determined by Hill curve fitting ( Figure A2). (b) EMSAs using operator mutant fragments. (c) In-gel Cu-OP footprinting using a 168 bp fragment covering the intergenic region and part of the ah-barR coding region, with the bottom strand being 32 P-labeled. A, T, G, and C represent the sequencing ladders, "−" corresponds to input DNA, and "+" corresponds to the Ah-BarR-DNA complex. Protected areas are indicated with a green vertical line, while the predicted binding sites are indicated with a gray box, and the TATA box/BRE elements are indicated with light/dark blue boxes, respectively. The protection/hyperreactivity zones are indicated on the DNA sequence. Nucleotides of the bottom strand are colored relative to the degree of protection (values range from 0.3 to 4.3). Predicted binding sites are indicated with a gray box, and the TATA box/BRE element with light/dark blue boxes, respectively. (d) Consensus sequence for Ah-BarR binding, based on the three identified binding sites TFBS 1, TFBS 2, and TFBS 3. F I G U R E 4 Effect of naringenin-induced Ah-BarR expression and addition of different β-alanine concentrations on mKate2 expression of strains BS1 and BS2, harboring Acidianus hospitalis promoters P barR and P at , respectively. Fluorescence/OD 600 is expressed in relative fluorescence units (RFU). Each point corresponds to the corrected mean relative fluorescence over three time points of four biological replicates, error bars indicate the standard deviation across four replicates. BS1, BS2, BS1/2 noTF, and Control are respectively colored in turquoise, dark blue, pink, and gray. Analysis of statistical significance is provided (Table A5). (a) Response of BS1 and BS2 to different naringenin concentrations (range 0-60 mg/L). *p < 0.05; **p < 0.01; ***p < 0.001 (calculated with one-way analysis of variance [ANOVA] and Tukey's HSD test) are indicated in comparison to the noTF 0 mg/L naringenin condition. The red asterisks indicate the significant decrease observed for the 60 mg/L condition (compared to the 15 mg/L naringenin condition) of BS2. (b) Response of BS1 and BS2 to different concentrations of β-alanine at a fixed concentration of 20 mg/L naringenin. Concentrations of β-alanine range from 0 to 10 mM. Responses of BS1 and BS2 to β-alanine are displayed on a symmetrical logarithmic x-axis along with their fit to a Hill function (see also Section 2.6).
F I G U R E 5 Effect of naringenin-induced Ah-BarR expression and addition of different β-alanine concentrations on the mKate2 expression of strains BS3 and BS4, harboring Sulfolobus acidocaldarius promoters P sa-barR and P sa-at , respectively. Fluorescence/OD 600 is expressed in relative fluorescence units (RFU). Each point corresponds to the corrected mean relative fluorescence over three time points of four biological replicates, error bars indicate the standard deviation across four replicates. BS3, BS4, BS3/4 noTF, and Control are respectively colored in turquoise, dark blue, pink, and gray. Analysis of statistical significance is provided (Table A5). (a) Response of BS3 and BS4 to different naringenin concentrations (range 0-60 mg/L). *p < 0.05; **p < 0.01; ***p < 0.001 (calculated with one-way analysis of variance [ANOVA] and Tukey's HSD test) are indicated in comparison to the noTF 0 mg/L naringenin condition. b. Response of BS3 and BS4 to different concentrations of β-alanine at a fixed concentration of 20 mg/L naringenin. Concentrations of β-alanine range from 0 to 10 mM. Responses of BS3 and BS4 to β-alanine are displayed on a symmetrical logarithmic x-axis along with their fit to a Hill function (see also Section 2.6).
is determined by other factors ( Figure A7). Surprisingly, the truncated mutant showed an increased sensitivity to β-alanine, with a threshold value of 0.26 mM compared to 0.37 mM for WT Ah-BarR (Figure 7, Table 2). Also, in terms of DNA-binding properties, the truncated Ah-BarR mutant acted differently as compared to WT Ah-BarR: two distinct complexes were observed in the absence of β-alanine, while complexes resided in the wells in the presence of β-alanine ( Figure A6).

| DISCUSSION
Our work demonstrates that Ah-BarR is a dual-function regulator that is capable of repressing transcription of the promoter of its own gene and of activating transcription of the promoter of a divergently located aminotransferase gene. This was revealed by monitoring the functional regulation of the archaeal regulator and its native promoter/operator region in E. coli. Based on these observations, it could be hypothesized that in A. hospitalis, Ah-BarR is capable of simultaneously performing repression and activation of each of the divergently oriented genes while being bound as an octamer to the intergenic region. However, this hypothesis is in contrast to the observation of Sa-BarR performing auto-activation in S. acidocaldarius (Liu et al., 2014). The F I G U R E 6 In silico docking of β-alanine in the ligand binding pocket of Ah-BarR. Docking was performed in AutoDock Vina for a SWISS-MODEL-generated structural model of Ah-BarR. Residues involved in the formation of the binding pocket are shown. β-alanine is depicted in green, and chains corresponding to different monomers are colored distinctly (blue vs. orange).
F I G U R E 7 Response of BS1 strains with Ah-BarR mutants to different concentrations of β-alanine at a fixed concentration of 20 mg/L naringenin. Concentrations of β-alanine range from 0 to 10 mM. Fluorescence/OD 600 is expressed in relative fluorescence units (RFU). Each point corresponds to the corrected mean relative fluorescence over three time points of four biological replicates, error bars indicate the standard deviation across four replicates. Responses of the BS1 mutants (M103A, M103N, M103T, T134A, T136A, and trunc) to β-alanine are displayed on a symmetrical logarithmic x-axis along with their fit to a Hill function (see also Section 2.6). BS1 mutant, BS1 noTF, and Control are respectively colored in turquoise, pink, and gray. Analysis of statistical significance is provided (Table A6). observation that in the E. coli reporter gene system, transcriptional repression was also observed for the barR promoter of S. acidocaldarius in combination with Ah-BarR, indicates that there are differences in regulatory response between the Ah-BarR system studied in an E. coli host and the native Sa-BarR system (Liu et al., 2014). This might be attributed to host-specific differences (e.g. the basal transcription machinery), or to differences between Ah-BarR and Sa-BarR functionalities, which is unlikely given their high level of sequence identity (Figure 1a).
Based on our data, we can therefore not conclude whether or not Ah-BarR is also a dual-function regulator in A. hospitalis.
It is however clear that the regulatory effect by Ah-BarR is achieved upon interacting with multiple binding sites in the intergenic region between barR and the aminotransferase target gene. Although a similar binding site organization is observed for Ah-BarR and its homologs Sa-BarR and St-BarR, there are small but significant differences (Figure 8a). Sa-BarR and St-BarR are predicted to interact with 4 to 5 regularly spaced sites each with spacer lengths between F I G U R E 8 Putative model of DNA binding and interaction with β-alanine of the transcription factor Ah-BarR in the order Sulfolobales. (a) Schematic overview and multiple sequence alignment of the barR-aminotransferase intergenic region in Acidianus hospitalis, Sulfolobus acidocaldarius, and Sulfurisphaera tokodaii. Confirmed binding sites are indicated with a gray box, predicted low affinity binding sites with a red/ orange box, and the TATA box/BRE element, respectively with light/dark blue boxes. Distances (in nucleotides) between the promoter elements are indicated in the figure, and the transcription starts sites are colored yellow. Conserved nucleotides are indicated with an *. (b) Schematic hypothesis of the interaction between A. hospitalis BarR, the barR-aminotransferase intergenic region, and β-alanine. Octameric Ah-BarR binds binding sites TFBS 1, TFBS 2, TFBS 3, and LTFBS 4 in the absence of β-alanine, leading to repression of the barR gene and activation of the aminotransferase gene. In the presence of β-alanine, BarR undergoes a conformational change, leading to a change in binding preferences: Ah-BarR now binds TFBS 1, TFBS 2, TFBS 3, and LTFBS 5, leading to a transcriptional derepression of the ah-barR gene and deactivation of the aminotransferase gene.
15 and 17 bp, corresponding to an alignment on the DNA double helix in which similar binding site residues are separated by two helical turns (Liu et al., 2014). The same number of binding sites can be observed or predicted for Ah-BarR but with differences in spacing.
In in vitro experiments, three binding sites were identified for Ah-BarR in between both promoters and two additional putative auxiliary sites (LTFBS 4 and LTFBS 5) can be predicted based on sequence similarity with the consensus binding motif and/or conservation with respect to the S. acidocaldarius and S. tokodaii Upon interaction with β-alanine, regulatory effects are relieved while the regulator remains bound to DNA, as is the case for Sa-BarR (Liu et al., 2016). It is hypothesized that Ah-BarR undergoes small conformational changes; possibly these changes alter the binding site interaction pattern, for example by releasing the binding site LTFBS4 downstream of the TATA box in P barR and binding an additional binding site LTFBS5, which is located at a distance of 17 bp from binding site TFBS3. As a result, interaction with the basal transcription machinery is altered, leading to a derepression of ah-bar transcription and deactivation of at transcription. Interesting differences were observed when comparing BarR to its homologs. More specifically, the very long C-terminal tail and the residue on the position corresponding to M103 were different in all homologs.
Mutagenesis studies have indicated that an Ah-BarR mutant without a tail (cfr. truncated mutant) is not only affected by how it interacts with DNA, but it also shows a more sensitive ligand response compared to wild-type BarR. The functional role of this longer Cterminal tail remains unclear (Ziegler & Freddolino, 2021), although an allosteric functioning can be proposed. On the other hand, mutational studies have proven that a complete loss of functionality of residue M103 (cfr. M103A mutant) leads to a less sensitive βalanine response, but that substitution with threonine or asparagine (as observed in Sa-BarR, St-BarR, and Grp) restores the sensitivity to the ligand β-alanine, and not to glutamine as was hypothesized.

CONFLICT OF INTEREST STATEMENT
None declared.

DATA AVAILABILITY STATEMENT
All data are provided in the results and appendix of this paper.

ETHICS STATEMENT
None required.         T A B L E A8 Statistical significance assessment performed to compare FL/OD 600 values obtained for the BS1 mutants grown in the presence of 0 or 5 mM β-alanine/glutamine. F I G U R E A1 DNA sequence of gb_Ahos. This 750 bp sequence is part of the Acidianus hospitalis genome and contains (parts of) the genes ahos_rs02205 (ah-barR) and ahos_rs02210 (aminotransferase gene) and the intergenic region. Predicted binding sites are indicated in gray.

ORCID
F I G U R E A2 Hill curve fitting enabling quantification of the binding of Ah-BarR WT or mutants to different DNA fragments. (a) Binding of WT Ah-BarR to the 274 nt native promoter fragment (corresponding to Figure 2a). (b) Binding of WT Ah-BarR to the 274 nt mutated promoter fragments (mut1, mut2, and mut3) (corresponding to Figure 2b). (c) Binding of BarR mutant proteins (M103A, M103N, M103T, T134A, T136A, and trunc) to the 274 nt native promoter fragment (corresponding to Figure A6b). (d) Binding of WT Ah-BarR to the 274 nt native promoter fragment (corresponding to Figure A6a). (e) Binding of WT Ah-BarR to the 168 nt native promoter fragment (corresponding to Figure A6a).

F I G U R E A4
Effect of amino acids on the expression level of BS1 and BS2. Fluorescence/OD 600 is expressed in relative fluorescence units (RFU). Each point corresponds to the corrected mean relative fluorescence over three timepoints of four biological replicates, error bars indicate the calculated standard deviation over the four replicates. BS1, BS2, BS1/2 noTF, and Control are respectively colored in light blue, dark blue, pink, and gray. A "−" corresponds to a concentration of 0 mg/L naringenin, while a "+" corresponds to a concentration of 20 mg/L naringenin. *p < 0.05; **p < 0.01; ***p < 0.001 (calculated with one-way analysis of variance [ANOVA] and Tukey's HSD test) are indicated in comparison to the "+" condition (Table A7). (a) Response of BS1 and BS2 to addition of eight different amino acid pools (amino acids added in concentration of 3/5 mM) to the medium. (b) Response of BS1 and BS2 to addition of amino acids from pool 1 and 7 (amino acids added in concentration of 3/5 mM) to the medium. F I G U R E A7 Comparison of β-alanine and glutamine response of Ah-BarR WT and mutants. Concentrations of 0 mM (corresponding to "−") or 5 mM β-alanine/glutamine are used, together with a fixed concentration of 20 mg/L naringenin. Fluorescence/OD 600 is expressed in relative fluorescence units (RFU). Each point corresponds to the corrected mean relative fluorescence over three timepoints of four biological replicates. ***p < 0.001 (calculated with one-way analysis of variance [ANOVA] and Tukey's HSD test) is indicated in comparison to the corresponding '−' condition (Table A8).